Allergens stimulate release of mast cell inflammatory mediators by crosslinking IgE bound to the high affinity IgE receptor, FcepsilonRI. By immunogold electron microscopy of native membrane sheets, we have recently shown that multiple, distinct microdomains mediate signaling and receptor internalization in mast cells. Receptors cluster together with Lyn in resting cells, are phosphorylated by Lyn very rapidly after stimulation in the signal initiation step, and then segregate from Lyn as receptors accumulate in osmiophilic regions of membrane. For signal propagation, these dark patches of membrane also accumulate Syk, PLCgamma2, p85, Gab2 and Grb2, identifying the osmiophilic patch as a primary site of FcepsilonRI signaling. Recruitment of AP-2, Eps15 and clathrin to receptors in osmiophilic patches, and the budding of clathrin-coated vesicles there, also define it as the primary site of FceRI internalization. Secondary signaling domains include p85 and PLCgamma1 and may be organized around the palmitoylated transmembrane adaptor protein, LAT. We hypothesize that these distinct microdomains perform separate tasks in mast cell signaling and receptor trafficking. To do so, they require unique lipid and protein compositions. We further propose that, after the initial segregation of receptors from Lyn, receptors go through a series of interaction and segregation steps with signaling and internalization machinery. The "segregation model" predicts that these successive association/dissociation steps are ordered in part by features of the osmiophilic patch. To test these hypotheses, we propose to use a combination of electron microscopy, recombinant protein expression, biochemical assays and mass spectrometry to map the distribution of proteins and lipids in primary and secondary domains. We will test roles for acylation or prenylation of key signaling proteins in their targeting to either primary or secondary domains. We will also define the requirement for ubiquitin ligases and multiubiquitination in receptor endocytosis. This work will define roles for microdomains in the processes of signal initiation, signal propagation and receptor internalization.